1. Field of the Invention
This invention relates to improved adjuvant and vaccine compositions, methods for preparing said improved adjuvant and vaccine compositions, and methods of using the improved compositions.
2. Description of the Prior Art
Conventional vaccines have been used for many years to protect humans and animals from a wide variety of infectious diseases. Typically, these conventional vaccines contain one or more antigens which may include an attenuated pathogen, killed pathogen, or an immunogenic component of a pathogen. In some vaccines, the antigen or antigens may be employed alone to elicit protective immune responses. In other vaccines, the antigen or antigens may be employed together with one or more adjuvants to enhance the immunogenicity of an antigen. One such adjuvant known to the art is monophosphoryl lipid A, which is derived from the lipopolysaccharide of Salmonella minnesota R595. It is also known to the art that monophosphoryl lipid A is a lipidic material which spontaneously aggregates with itself in an aqueous environment. Moreover, it is known that the degree of aggregation has an effect on the activity of monophosphoryl lipid A as an immunostimulant in that the aggregated monophosphoryl lipid A is less stimulatory.
Monophosphoryl lipid A is typically obtained as the triethylamine salt in the form of a lyophilized white powder. Being very hydrophobic, the lyophilized monophosphoryl lipid A does not readily form a clear solution when reconstituted with water but instead yields a turbid mixture with visible white particulates of heterogeneous size that settle out and further aggregate upon standing. To make an acceptable aqueous preparation of monophosphoryl lipid A, it is known to suspend the lyophilized monophosphoryl lipid A triethylamine salt at 1 to 2 mg/mL (w/v) in water containing 0.2% triethylamine, to heat the suspension at 65-70xc2x0 C., and then to sonicate the mixture. The resulting aqueous preparation, slightly opalescent or clear, is an aqueous colloidal suspension. The triethylamine aids in the solubilization of the monophosphoryl lipid A and may be substituted with similar amounts of triethanolamine.
When aqueous preparations of monophosphoryl lipid A prepared as described hereinabove are frozen and then thawed, however, the monophosphoryl lipid A aggregates resulting in a turbid mixture quite similar in appearance to the turbid mixture of monophosphoryl lipid A prior to sonication. Similarly, when an aqueous preparation of monophosphoryl lipid A as described hereinabove is lyophilized and then rehydrated, the result is also a turbid mixture of aggregated monophosphoryl lipid A.
The present invention provides to the art a lyophilized composition containing monophosphoryl lipid A, which composition exhibits an enhanced reconstitution feature and which avoids the settling out and aggregation problems of the prior art. In particular, the present invention provides a lyophilized composition comprising monophosphoryl lipid A, sugar and, optionally, an added amine based surfactant, and is capable of being reconstituted or rehydrated with an aqueous diluent to form, without further sonication, an aqueous colloidal suspension of monophosphoryl lipid A having a light transmission of at least 88%, as measured spectrophotometrically. The lyophilized composition according to the present invention comprises up to about 5 wt % monophosphoryl lipid A, greater than about 70 wt % sugar and from about 0 to about 30 wt % optionally added amine based surfactant, said wt % based on the total of the weights of monophosphoryl lipid A, sugar and, if present, amine based surfactant. Preferably, the lyophilized composition according to the present invention comprises up to about 5 wt % monophosphoryl lipid A, from about 70 to about 99.99 wt % sugar and from about 0 to about 28 wt % optionally added amine based surfactant. More preferably, the lyophilized composition according to the present invention comprises up to about 4 wt % monophosphoryl lipid A, from about 75 to about 99.99 wt % sugar and from about 0 to about 22 wt % optionally added amine based surfactant. The lyophilized composition may further comprise an immunologically effective amount of an antigen or antigens. The lyophilized composition of the present invention may be reconstituted or rehydrated with an aqueous diluent at concentrations up to about 210 mg of lyophilized composition per ml of aqueous diluent, preferably from about 10 mg of lyophilized composition per ml of aqueous diluent to about 210 mg of lyophilized composition per ml of aqueous diluent, to form, without further sonication, an aqueous colloidal suspension.
Another aspect of the present invention is a method of preparing an aqueous colloidal suspension of monophosphoryl lipid A in which the aqueous colloidal suspension is frozen for storage and then thawed for use without the problems of settling out and aggregation known in the prior art. By this method, monophosphoryl lipid A is mixed in an aqueous diluent and optionally with an amine based surfactant and also optionally an antigen or antigens. An aqueous colloidal suspension is formed by sonicating, optionally with heating, or other known methods, as described in greater detail hereinafter. Sugar, in an amount from about 10 mg/ml to about 200 mg/ml, is added to the mixture either before or after the formation of an aqueous colloidal suspension. The sugar may be in the form of a solid or in the form of an aqueous solution. The resulting aqueous colloidal suspension may then be frozen. Thawing the frozen aqueous colloidal suspension affords without further sonication an aqueous colloidal suspension containing monophosphoryl lipid A having a light transmission of greater than or equal to 88%, as measured spectrophotometrically. An antigen or antigens, as defined hereinafter, may be added to the thawed aqueous colloidal suspension to form a vaccine composition which may be administered to a vertebrate. Alternatively, if the aqueous colloidal suspension contains an antigen before freezing, the vaccine composition may be thawed and administered to a vertebrate.
The aqueous colloidal suspensions of the present invention are a special type of liquid suspension in which the particles of suspended monophosphoryl lipid A are present in very finely divided but not in dissolved form. The aqueous colloidal suspensions containing monophosporyl lipid A, sugar and, optionally, an amine based surfactant according to the present invention are true suspensions not solutions, and do not have the property, unlike ordinary suspensions of monophosphoryl lipid A, of settling out and aggregation. The presence of the aqueous colloidal suspensions of the present invention can be determined by light transmission. Thus, an aqueous colloidal suspension containing monophosphoryl lipid A, sugar and optionally an amine based surfactant according to the present invention is one which exhibits a light transmission of greater than or equal to 88%, as measured spectrophotometrically.
The present invention solves the settling out and aggregation problems of the prior art, by providing the addition of sugar to an aqueous colloidal suspension of monophosphoryl lipid A prior to freezing or lyophilization. The sugar may be added either before or after formation of the aqueous colloidal suspension but must be added before freezing or lyophilization of the suspension. The addition of sugar to an aqueous colloidal suspension of monophosphoryl lipid A prior to freezing or lyophilization provides a composition which, after freezing can be thawed to afford an aqueous colloidal suspension without further sonication or, alternatively, after lyophilization, can be reconstituted with a suitable aqueous diluent and afford without further sonication an aqueous colloidal suspension as described hereinabove. Suitable sugars include the monosaccharides, dextrose, mannose, galactose and fructose as well as the disaccharides sucrose, lactose, isomaltose, maltose and trehalose. Mixtures of sugars, for example sucrose and dextrose, may also be employed. These sugars are all non toxic and pharmaceutically acceptable. Preferred are sucrose and dextrose. The sugar may be in the form of a solid or in the form of an aqueous solution. Suitable aqueous diluents include water or saline and can also include an antigen or antigens and, may additionally contain preservatives or additional adjuvants, or other pharmaceutically acceptable additives, vehicles, or carriers. Suitable amine based surfactants include triethylamine (TEA) and triethanolamine (TEM).
A further aspect of the invention is a reconstituted or rehydrated aqueous colloidal suspension which, despite the elimination of a further sonication step, is obtained upon reconstitution of the lyophilized composition described hereinabove with an aqueous diluent. As discussed hereinabove, before the present invention, a sonication step was necessary in order to obtain an aqueous colloidal suspension containing monophosphoryl lipid A. However, it has now been found that when an aqueous diluent is added to the lyophilized composition described hereinabove, an aqueous colloidal suspension containing monophosphoryl lipid A is obtained without further sonication. The reconstituted aqueous colloidal suspension so obtained exhibits a light transmission of greater than or equal to 88%, when measured spectrophotometrically. Surprisingly, the reconstituted aqueous colloidal suspension so obtained is capable of being frozen and, after thawing, again reforming an aqueous colloidal suspension which exhibits a light transmission of greater than or equal to 88%. The reconstituted aqueous colloidal suspension of the present invention comprises up to about 2.5 mg of monophosphoryl lipid A per ml of aqueous diluent, from about 10 to 200 mg of sugar per ml of aqueous diluent, and from about 0 to about 6 mg of amine based surfactant per ml of aqueous diluent. Preferably, the reconstituted aqueous colloidal suspension of the present invention comprises up to about 2.0 mg of monophosphoryl lipid A per ml of aqueous diluent, from about 20 to 150 mg of sugar per ml of aqueous diluent and from about 0 to about 3 mg of amine based surfactant per ml of diluent. The reconstituted aqueous colloidal suspension may further comprise an immunologically effective amount of an antigen or antigens. Suitable sugars, amine based surfactants and aqueous diluents are as described hereinabove.
A further aspect of the invention is a vaccine composition comprising the lyophilized composition and the reconstituted aqueous colloidal suspension described hereinabove in combination with an immunologically effective amount of an antigen or antigens. The effective amount of an antigen or antigens may be optionally provided in the aqueous diluent. In particular, the vaccine composition further comprises an immunologically effective amount of an antigen or antigens derived from or produced by a bacterium, a virus, a parasite, a cancer cell or an allergen. An effective amount of antigen is defined as that amount of antigen that when administered to an animal or a human evokes an immune response as measured by production of specific antibodies or cell-mediated effector mechanisms. Immunologically effective amounts of an antigen or antigens are in general from about 1 xcexcg or less to 5 mg. An effective amount of the monophosphoryl lipid A adjuvant is the amount of monophosphoryl lipid A that when added to a vaccine will enhance the magnitude or quality or duration of the immune response to the antigen or antigens in the vaccine. An effective amount of the adjuvant monophosphoryl lipid A is in the range of about 1 xcexcg to about 1 mg.
Suitable antigens for the vaccine compositions of the present invention include any entity capable of producing an antibody or cell-mediated immunological response directed specifically against that entity in a vertebrate exposed to the antigen. One or more antigens may be employed. The antigen or antigens may be derived from pathogenic micro-organisms including viruses, bacteria, mycoplasmas, fungi, protozoa and other parasites. Further, the antigen or antigens may be derived from sources other than microorganisms, for example, cancer cells or allergens. The antigen or antigens may be all or part of a pathogenic microorganism, or all or part of a protein, glycoprotein, glycolipid, polysaccharide or lipopoly-saccharide which is associated with the organism, or the antigen or antigens may be a polypeptide or other entity which mimics all or part of such a protein, glycoprotein, glycolipid, polysaccharide or lipopolysaccharide.
Pathogenic microorganisms from which antigens may be produced or derived for vaccine purposes are well known in the field of infectious diseases, as listed in, for example, Medical Microbiology, Second Edition, (1990) J. C. Sherris (ed.), Elsevier Science Publishing Co., Inc., New York, and Zinsser Microbiology, 20th Edition (1992), W. K. Joklik et al. (eds.), Appleton and Lange Publishing Division of Prentice Hall, Englewood Cliffs, N.J. Examples of organisms of interest for human vaccines include Chlamydia, Nontypeable Haemophilus influenzae, Helicobacter pylori, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonella typhi, Streptococcus pneumoniae, Group A Streptococcus, Group B Streptococcus, Herpes Simplex Virus, Human Immunodeficiency Virus, Human Papilloma Virus, Influenza, Measles, Parainfluenza, Respiratory Syncytial Virus, Rotavirus, Norwalk Virus, and others.
The antigen or antigens may include glycoconjugates which comprise polysaccharide antigen or antigens, for example, bacterial capsular polysaccharide or fragment thereof, chemically linked to a protein carrier molecule in order to enhance immunogenicity. Methods for preparing conjugates of bacterial capsular polysaccharide and protein carrier molecules are well known in the art and can be found, for example, in Dick and Burret, Contrib Microbiol Immunol. 10:48-114(Cruse J M, Lewis R E Jr., eds; Basel Kruger (1989). Suitable conjugates, including pneumococcal glycoconjugate, are described in greater detail in U.S. Pat. Nos. 4,673,574, 4,761,283, 4,902,506, 5,097,020 and 5,360,897 the contents of which are incorporated herein by reference.
Also provided is a method of immunizing a vertebrate through vaccination which comprises administrating an effective amount of a vaccine composition according to the present invention to said vertebrate.
Also provided is a method for the preparation of a lyophilized composition comprising:
a. suspending monophosphoryl lipid A in an amount up to about 5 mg/ml and, optionally, an amine based surfactant in an amount from 0 to about 6 mg/ml in an aqueous diluent;
b. forming an aqueous colloidal suspension having a light transmission of greater than or equal to 88%, as measured spectrophotometrically;
c. adding sugar at about 10 to 200 mg/ml either before or after forming the aqueous colloidal suspension;
d. lyophilizing the sugar containing aqueous colloidal suspension; and
e. recovering a lyophilized composition.
Also provided is a method for preparing a lyophillized composition comprising:
a. heating lipopolysaccharide of gram negative bacteria Salmonella minnesota R595 in a mineral acid of moderate strength for a sufficient period of time to obtain a monophosphoryl derivative;
b. dissolving the monophosphoryl derivative in an organic solvent and drying;
c. treating the monophosphoryl derivative with mild alkali to remove a base labile fatty acid chain at the position to yield 3-deacylated monophosphoryl lipid A;
d. purifying the 3-deacylated monophosphoryl lipid A by liquid chromatography and recovering monophosphoryl lipid A;
e. suspending monophosphoryl lipid A in an amount up to about 5 mg/ml and, optionally, an amine based surfactant in an amount from 0 to about 6 mg/ml in an aqueous diluent;
f. forming an aqueous colloidal suspension having a light transmission of greater than or equal to 88%, as measured spectrophotometrically;
g. adding sugar at about 10 to 200 mg/ml either before or after forming the aqueous colloidal suspension;
h. lyophilizing the sugar containing aqueous colloidal suspension; and
i. recovering a lyophilized composition.
Also provided is a method for the preparation of an aqueous colloidal suspension containing monophosphoryl lipid A capable of being frozen and thawed comprising:
a. suspending monophosphoryl lipid A in an amount up to about 5 mg/ml and, optionally, an amine based surfactant in an amount from 0 to about 6 mg/ml in an aqueous diluent;
b. forming an aqueous colloidal suspension having a light transmission of greater than or equal to 88%, as measured spectrophotometrically;
c. adding sugar at about 10 to 200 mg/ml either before or after forming the aqueous colloidal suspension;
d. freezing the sugar containing aqueous colloidal suspension; and
e. thawing and recovering the aqueous colloidal suspension.
The preparation of monophosphoryl lipid A is described in U.S. Pat. No. 4,912,094, the contents of which are incorporated herein by reference. Briefly, monophosphoryl lipid A is produced by refluxing lipopolysaccharide (or lipid A) obtained from heptoseless mutants of gram negative bacteria, Salmonella minnesota R595, in mineral acid solutions of moderate strength (e.g., 0.1N HCl) for a period of approximately 30 minutes. Suitable mineral acids include hydrochloric, sulfuric and the like. This treatment results in the loss of the phosphate moiety at position 1 of the reducing-end glucosamine. The core carbohydrate is removed from the 6xe2x80x2 position of the non-reducing glucosamine during this treatment. The result is a monophosphoryl derivative of lipid A. The monophosphoryl derivative of lipid A is dissolved in organic solvents and treated with very mild alkali which removes the base-labile fatty acid chain at the 3 position to yield 3-O-desacyl-4xe2x80x2-monophosphoryl lipid A, indicating that position 3 of the reducing end glucosamine is de-O-acylated. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. Suitable organic solvents include methanol (alcohols), dimethyl sulfoxide, dimethylformamide, chloroform, dichloromethane and the like as well as mixtures thereof. Combinations of water and one or more of these organic solvents also can be employed. Suitable alkaline bases can be chosen from among various hydroxides, carbonates, phosphates and amines. Illustrative bases include the inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and the like, and organic bases such as alkyl amines and include, but are not limited to, diethylamine, triethylamine and the like. The 3-O-desacyl-4xe2x80x2-monophosphoryl lipid A is purified by liquid chromatography and converted to the monobasic triethylamine (triethylammonium) salt.
The term monophosphoryl lipid A as used herein means 3-O-desacyl-4xe2x80x2-monophosphoryl lipid A as the monobasic triethylamine (triethylammonium)salt.
To prepare the lyophilized composition of the present invention, the monophosphoryl lipid A is added to an aqueous diluent, preferably water, in amounts up to 5 mg of monophosphoryl lipid A per ml of aqueous diluent, preferably up to 2.5 mg/ml and more preferably from about 0.5 to 2.5 mg/ml. Optionally, an added amine based surfactetnt in an amount from about 0 to about 6 mg/ml, preferably 0 to 3 mg/ml is employed.
An aqueous colloidal suspension having a light transmission of greater than or equal to 88%, as measured spectrophotometrically is formed by sonication, optionally with heating, or other methods. Heating is optional but preferred to facilitate the formation of the aqueous colloidal suspension of monophosphoryl lipid A. Suitable sonication equipment include, for example, a probe sonicator (Vibracell VCX600; Sonica) attached to probes whose sizes are appropriate for the volume being processed or a bath sonicator such as the Model No. G112SP1T obtained from Laboratory Supplies Co. Inc., (Hicksville, N.Y.). Other similar equipment used in the pharmaceutical industry would also be appropriate for sonication of monophosphoryl lipid A.
The aqueous colloidal suspension of monophosphoryl lipid A may be formed by methods other than sonication, for example, by shearing forces as would be generated in a microfluidizer.
Sugar is also added either before or after formation of the aqueous colloidal suspension, in amounts from 10 to 200 mg sugar per ml of aqueous diluent, preferably from about 20 to 150 mg/ml. The aqueous colloidal suspension, containing monophosphoryl lipid A, sugar and optionally an added amine based surfactant and optionally an immunologically effective amount of an antigen or antigens in the amounts recited hereinabove, is lyophilized to afford the lyophilized composition according to the present invention.
The aqueous colloidal suspension of monophosphoryl lipid A, sugar and, optionally, an amine based surfactant of an antigen or antigens is lyophilized to afford the lyophilized composition of the present invention. As is known to those skilled in the art, lyophilization is a process of drying in which water is sublimed from the product after it is frozen, by applying a vacuum. Specifics of lyophilizing or freeze-drying are described in Remington""s Pharmaceutical Sciences. Chapter 84, page 1565, 18th Edition, A. R. Gennaro, Editor, 1990, Mack Publishing Company.
Whether an aqueous colloidal suspension is formed is determined by measuring the light transmission. It has been found that compositions having a light transmission of at least 88% exhibit the properties of colloidal suspensions. Light transmission is measured using a spectrophotometer in which is illuminated a liquid sample in a glass, quartz or plastic cuvette with a light path of 1 centimeter. The light may be in the visible or invisible spectrum, but for measurements of light transmission of this type a wavelength of 650 nm may appropriately be used. The amount of light passing through the sample (i.e. transmitted) is referenced to a blank cuvette containing the solvent or diluent in which the material is dissolved or suspended. Samples that do not absorb or scatter the light will exhibit 100% light transmission whereas those that absorb or scatter all the light will have 0% light transmission.
While not wishing to be bound by theory, it is believed that the advantageous results of the invention are obtained because the addition of sugar either before or after the formation of an aqueous colloidal suspension containing monophosphoryl lipid A prevents the monophosphoryl lipid A from aggregation either upon freezing or thawing of the aqueous colloidal suspension or upon lyophilizing the aqueous colloidal suspension and reconstitution or rehydration with an aqueous diluent. By including sugar in an aqueous colloidal suspension containing monophosphoryl lipid A prior to lyophilization, the lyophilized composition can be reconstituted with an aqueous diluent such as water or saline without the problem of reaggregation of the monophosphoryl lipid A. In addition, freezing of the reconstituted colloidal suspension or vaccine composition does not cause aggregation to reoccur. Similarly, by including sugar in an aqueous colloidal suspension containing monophosphoryl lipid A prior to freezing, upon thawing a frozen aqueous colloidal suspension is again obtained without the need for further sonication. The ability of sugar to prevent aggregation of the monophosphoryl lipid A is evident regardless of whether the aqueous colloidal suspension containing monophosphoryl lipid A is prepared in water alone, or in water containing triethylamine or triethanolamine.
Thus, the addition of sugar to monophosphoryl lipid A containing aqueous compositions, either before or after forming an aqueous colloidal suspension, provides surprising and unexpected results when such aqueous colloidal suspensions are either frozen and thawed or lyophilized and reconstituted. Such results further permit the advantageous preparation of vaccine compositions.
The following examples are provided to illustrate the invention.